(A) Immunoblotting of lysates from MCF10A cells stably expressing flag-tagged E4 ORF1, ORF2, ORF3, ORF4, or ORF6. The top panel was probed with FLAG antibody, and the bottom panel was probed with tubulin antibody to control for loading. (B) Glucose consumption and lactate production rates from MCF10A cells stably expressing the flag-tagged ORFs indicated in (A). (C) Immunoblotting of lysates from MCF10A cells stably expressing HA-tagged E4ORF6 along with an inducible empty vector, flag-tagged ORF1 WT, or flag-tagged ORF1ΔPDZ. Briefly, the cells were treated with 1ug ml−1 doxycycline for 24 hours and subsequently starved for 24 hours in serum-free media with 1ug ml−1 doxycycline. To test the activation of PI3K, lysates were probed for phospho-Ser473-AKT; total AKT was used as a loading control, and FLAG was used to indicate induced ORF1 expression. (D) Cells described in (C) were induced to express flag-tagged ORF1 WT or flag-tagged ORF1ΔPDZ, and glucose consumption and lactate production rates were measured in full serum conditions. Error bars denote standard errors of the mean (n=3). * denotes p<0.05; ** denotes p<0.01.